Neospora caninum isolate

ABSTRACT

The present invention relates to a novel  Neospora caninum  isolate from Nowra and extracts thereof. The strain is useful in the development of diagnostic assays for the detection of parasites in animals. The present invention also relates to pharmaceutical compositions, using live or killed organisms or extracts thereof, for the treatment and prevention of parasitic infections in animals.

FIELD OF THE INVENTION

The present invention relates to a novel parasitic protozoan isolate andextracts thereof. The strain is useful in the development of diagnosticassays for the detection of parasites in animals. The present inventionalso relates to pharmaceutical compositions, using live organisms orextracts thereof, for the treatment and prevention of parasiticinfections in animals.

BACKGROUND OF THE INVENTION

The Apicomplexa is a diverse phylum of protozoa containing some of themost pathogenic organisms known to man. Representatives include taxa ofthe well known genera such as Plasmodium, Babesia and Toxoplasma. In1984 a research group in Norway reported a previously unknowncyst-forming protozoan which caused encephalitis and myositis in dogs(Bjerkas et al. 1984). These clinical signs were consistent with thosecaused by Toxoplasma gondii, a widely distributed member of theApicomplexa. However, unlike T. gondii, the organism was not pathogenicto outbred mice, nor were T. gondii specific antibodies present in thesera of the dogs studied. This prompted further investigation whichdemonstrated conclusively the organism was ultrastructurally andantigenically distinct from T. gondii (Dubey et al 1988). Neosporacaninum was thus described as a new member of the Sarcocystidae. Soonafter, N. caninum was implicated as a cause of abortion in cattle(Thilstead & Dubey, 1989).

An increasing number of N. caninum isolates have now been obtained,using techniques involving predominantly the direct inoculation ofinfected tissues onto tissue culture cells (Dubey et al. 1988, Conrad etal. 1993), although bovine isolates have proven difficult to obtain. Theprimary reason for this has been the detrimental effects of autolysis onthe viability of N. caninum in aborted bovine fetal tissues. Theisolation of a parasite population from a live animal is thereforepreferred.

Recent studies on the biological properties of bovine and canineisolates have suggested they belong to a single species called N.caninum (Holmdahl et al. 1997), despite the record that wide differencesexist between isolates in their biological properties. These includedifferences in antigenicity, ultrastructure, pathogenicity and geneticheterogeneity (Conrad et al. 1993, Marsh et al. 1995, Lindsay et al.1995 and Atkinson et al. 1999). Thus not all isolates of N. caninum maypossess the same properties, and indeed at least one isolate of N.caninum was mistakenly identified as Hammondia heydorni previously(Schares et al. 2001). Indeed, others have speculated on whether N.caninum and H. heydorni are the same or different species (Mehlhorn andHeydorn, 2000). Hammondia heydorni is also a cyst-forming coccidian thathas a life cycle which is very similar to that of N. caninum. Thus thetrue identity of the species N. caninum is currently being debated, andits relationship to H. heydorni is unclear.

In cattle, abortion due to N. caninum infections usually occur in mid-to late gestation, although not all infected foetuses are aborted. Manycongenitally infected calves are born healthy and persistently infected,although some infected calves are diseased at birth and die in theneonatal period with lesions similar to those of aborted calves.

The development and use of serological tests for the diagnosis ofneosporosis in livestock, along with the identification of animalsinfected or exposed to N. caninum, has been reviewed previously in greatdetail (Bjorkman et al. 1999; Atkinson et al. 2000a). Significantly,however, there is no effective vaccine against transplacentaltransmission or foetal loss which occurs as a result of neosporosis andattempts to formulate a vaccine have met with limited success.

Liddel et al. (1999) injected female BALB/c mice with a crude N. caninumtachyzoite lysate preparation co-administered with ImmuMAXSR™ adjuvant.These mice were subsequently mated, and pregnant dams were challengedwith N. caninum tachyzoites at 10-12 days of gestation. Results showed asingle injection offered complete protection against transplacentaltransmission of the parasite to the pups. All pups in this experimentalgroup were free from parasitic infection. No results have yet beenreported on the efficacy of this vaccine formulation in the bovine.

Baszler et al. (2000) examined the possibility of vaccination of BALB/cmice with soluble N. caninum antigen formulated in either nonionicsurfactant vesicles or Freunds Complete Adjuvant. This approach resultedin exacerbation of encephalitis and neurological disease in these mice.These observations were characterised by increased antigen specific IL-4secretion and increased IgG1:IgG2a ratios in vivo.

Adrianarivo et al. (1999) tested four different adjuvants with a killedwhole N. caninum tachyzoite preparation for immunogenicity. The resultsindicated that the immune responses, as determined by IFAT titres, weresignificantly higher in experimentally infected cattle compared toimmunised cattle.

Adrianarivo et al. (2000) studied the effect of a killed N. caninumtachyzoite preparation in pregnant cattle using a POLYGEN™ adjuvant.Heifers were injected at day 35 and day 65 of gestation and four weekslater were challenged with intravenous or intramuscular injection oftachyzoites. Post immunisation, heifers developed both humoral and cellmediated immune responses characterised by an increase in production ofIgG1 and IFN-γ respectively. Following a challenge with N. caninumtachyzoites, however, significant cell mediated immune response did notoccur. All foetuses in the study, both from control and experimentalcattle, developed lesions characteristic of N. caninum infection.Failure to prevent foetal infection by this formulation in pregnantcattle was concluded.

Unlike the development of killed or genetically engineered vaccinesagainst parasites, vaccines based on live populations of parasites areavailable, for example against Toxoplasma-induced abortion in sheep(Buxton & Innes, 1995) and Eimeria parasites of poultry (Shirley &Bedrnik, 1997). A live vaccine is not, however, available against N.caninum.

The literature on live vaccines against N. caninum is limited. Atkinsonet al. (1999) showed that infection of naive mice by the Nc-SweB1isolate of N. caninum partially protected them against a severeinfection by Nc-Liverpool. Lindsay et al. (1999) generated temperaturesensitive mutants of N. caninum and demonstrated that they could preventclinical signs associated with neosporosis in mice.

SUMMARY OF INVENTION

The present inventors have isolated a novel protozoan parasite from thecentral nervous system of a dairy calf. The parasite was identifiedbased on a number of criteria as an isolate of Neospora caninum and wascalled the “Nc-Nowra” strain. This isolate is naturally attenuated inits ability to cause neosporosis in a laboratory animal, and thus is anideal isolate to serve as a basis for a vaccine against this disease.

A sample of the Nc-Nowra isolate was deposited under the provisions ofthe Budapest Treaty on Jun. 21, 2001 with the Australian GovernmentAnalytical Laboratories (AGAL) and accorded AGAL Accession NoNM01/22338. Further, a sample of Vero cells was deposited under theprovisions of the Budapest Treaty on Jun. 21, 2001 with the AustralianGovernment Analytical Laboratories (AGAL) and accorded AGAL Accession NoNM01/22339.

Accordingly, in a first aspect the present invention provides aparasitic protozoan isolate having the characteristics of the isolatedeposited as AGAL Accession No. NM01/22338.

In a preferred embodiment of the first aspect, the isolate is thatdeposited as AGAL Accession No. NM01/22338.

In a second aspect, the present invention provides an antibody raisedagainst an isolate according to the first aspect. Preferably, theantibody is a monoclonal antibody.

In a third aspect the present invention provides a host cell infectedwith an isolate of the first aspect. Preferably, the host cell isderived from the sample deposited as AGAL Accession No NM01/22339.

In a fourth aspect, the invention provides a vaccine compositioncomprising an isolate of the first aspect, wherein the isolate is in theform of a lolled parasite population or live attenuated parasites.

The present invention also provides a vaccine composition comprising anextract or an isolate according to the first aspect. Preferably, theextract is selected from the group consisting of live attenuated, killedand fixed parasites, a cell lysate, an antigenic polypeptide and apolynucleotide encoding an antigenic polypeptide.

In a fifth aspect, the present invention provides a method for thetreatment or prevention of infection or disease in an animal, the methodcomprising administering to the animal a vaccine composition accordingto the fourth aspect.

In a sixth aspect, the present invention provides a method for thetreatment or prevention of infection or disease in an animal, the methodcomprising administering to the animal an antibody according to thesecond aspect.

In a seventh aspect, the present invention provides a method ofdiagnosing a parasitic infection or disease in an animal, the methodcomprising identifying the presence of the isolate according to thefirst aspect of the invention.

In a preferred embodiment of the fifth, sixth or seventh aspects, theparasitic disease is neosporosis. In a further preferred embodiment theparasitic infection is due to the presence of Neospora caninum. In oneembodiment the Neospora caninum is the isolate of the first aspect.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Scatterplot showing sera ELISA readings against N. caninum forindividual cows of the study herd.

FIG. 2. Bar chart showing IgG responses (as ELISA absorbances) to N.caninum antigen in mice infected with or without Nc-Nowra beforepregnancy. Groups given Nc-Liverpool at day 8 of pregnancy areindicated. Only mice receiving Nc-Nowra before pregnancy gave high IgGtitres.

DETAILED DESCRIPTION OF THE INVENTION

The parasitic isolate of the present invention may be used to developdiagnostic tools or aids for the detection or diagnosis of parasiticdisease in animals. Such diagnostic tools or aids include antigenicpolypeptides derived from the isolate, antibodies raised against theisolate and molecular probes or primers derived from the genome of theisolate.

Standard protein purification techniques can be used to isolateantigenic polypeptides from the novel parasitic strain of the presentinvention. Such techniques include selective precipitation with suchsubstances as ammonium sulphate, column chromatography,immunopurification methods, and the like. See, for instance, R. Scopes,Protein Purification: Principles and Practice, Springer-Verlag: New York(1982). Proteins and portions thereof isolated from the strain can besequenced according to standard techniques as described for instance inSambrook et al., Molecular Cloning, A Laboratory Manual, Cold SpringHarbour Publish., Cold Spring Harbour, N.Y. 2nd Ed (1989).

Antibodies raised against the strain may be polyclonal or monoclonalantibodies.

If polyclonal antibodies are desired, a selected mammal (e.g., mouse,rabbit, goat, horse, etc.) may be immunised with an immunogen preferablya purified protein mixed with an adjuvant. Serum from the immunisedanimal is collected and treated according to known procedures. Furtherfractionation of the antisera to enrich for antibodies reactive to theisolated proteins of the invention can be done if desired.

Monoclonal antibodies may be obtained by various techniques by oneskilled in the art Briefly, spleen cells in an animal immunised with adesired immunogen are immortalised, commonly by fusion with a myelomacell (See, Koller & Milstein, Eur. J. Immunol. 6:511-519 (1976)).Alternative methods of immortalization include transformation withEpstein Barr Virus, oncogenes, or retroviruses, or other methods wellknown in the art. Colonies arising from single immortalised cells arescreened for production of antibodies of the desired specificity andaffinity for the desired antigen.

Antibodies, both monoclonal and polyclonal, which are directed againstepitopes are particularly useful in diagnosis, and those which areneutralising are useful in passive immunotherapy. Monoclonal antibodies,in particular, may be used to raise anti-idiotype antibodies.Anti-idiotype antibodies are immunoglobulins which carry an “internalimage” of the antigen of the agent against which protection is desired.

Techniques for raising anti-idiotype antibodies are known in the art.These anti-idiotype antibodies may also be useful in therapy.

For the purposes of this invention, the term “antibody”, unlessspecified to the contrary, includes fragments of whole antibodies whichretain their binding activity for a target antigen. Such fragmentsinclude Fv, F(ab′) and F(ab′)₂ fragments, as well as single chainantibodies (scFv).

In one embodiment, the antibodies of the present invention bindspecifically to the Neospora isolate of the present invention.

Antibodies of the invention may be bound to a solid support and/orpackaged into kits in a suitable container along with suitable reagents,controls, instructions and the like.

Molecular probes or primers for detecting the isolate of the presentinvention may be generated by recombinant or synthetic means.Preferably, the polynucleotide is at least 16 nucleotides in length,more preferably at least 20, 25, 30 or 40 nucleotides in length. Thepolynucleotide may be used to produce a primer, e.g. a PCR primer, aprimer for an alternative amplification reaction, or a probe e.g.conjugated by conventional means to a radioactive or non-radioactivelabel.

Diagnosis may be achieved by detecting the presence of the parasite orby assaying for antibodies to the parasite in the animal. As there isnow concern that humans are susceptible to these type of parasiteinfections, the present invention includes human applications.

The parasite may be detected by culturing the parasite from a clinicalspecimen and identifying the parasite by microscopy, or may beidentified by the use of antibodies to the parasite or by detecting aportion of the genome of the parasite by molecular biologicaltechniques. The clinical specimen may be a biopsy, stool specimen, bloodsample, foetal tissue or the like. It will be appreciated that thediscovery of the parasite and its association with parasitic diseasewill allow its detection by any of the known methods of the art. It willalso be appreciated that molecular detection methods like polymerasechain reaction (PCR) can also be used to identify the presence of theparasite in a clinical specimen.

Immunological and immunoassay procedures in general, are described inBasic And Clinical Immunology 7th Ed. (D. Stites and A. Terr ed.) 1991.According to the present invention immunoassays may by performed in anyof several configurations, which are reviewed extensively in EnzymeImmunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Fla. (1980);Practice and Theory of Enzyme Immunoassays,” P. Tijssen, LaboratoryTechniques In Biochemistry And Molecular Biology, Elsevier SciencePublishers B. V. Amsterdam (1985). For example, a person skilled in theart would understand that the proteins and antibodies according to thepresent invention can be used in ELISA, immunoblot analysis andagglutination assays.

In brief, immunoassays to measure antibodies or antigens are eithercompetitive or noncompetitive binding assays. In competitive bindingassays, the sample analyte (e.g., anti-N. caninum antibodies) competeswith a labelled analyte (e.g., anti-N. caninum monoclonal antibody) forspecific binding sites on a capture agent (e.g., isolated N. caninumprotein) bound to a solid surface. The concentration of labelled analytebound to the capture agent is inversely proportional to the amount offree analyte present in the sample.

Noncompetitive assays are typically sandwich assays, in which the sampleanalyte is bound between two analyte-specific binding reagents. One ofthe binding agents is used as a capture agent and is bound to a solidsurface. The second binding agent is labelled and is used to measure ordetect the resultant complex by visual or instrument means.

Alternatively, the immunoassay is carried out in liquid phase and avariety of separation methods are employed to separate the boundlabelled component from the unbound labelled components. These methodsare known to those skilled in the art and include, but are not limitedto, immunoprecipitation, column chromatography, adsorption, addition ofmagnetisable particles coated with a binding agent and other similarprocedures.

In another alternate procedure, an immunoassay is carried out in liquidphase without a separation procedure. Typically, in these procedures,the assayed protein or other analyte competes with a compound forbinding to an antibody attached to a label. The binding of the analyteto an antibody, but not the binding of the compound to the antibody,causes a change in the signal emitted by the label, so that analytebinding is measured without separating the bound from the unboundlabelled component.

Western blot (immunoblot) analysis is also used to detect the presenceof antibodies to the parasitic isolate in a biological sample. Thistechnique is a reliable method for confirming the presence of antibodiesagainst a particular protein in the sample.

Pharmaceutical compositions prepared using extracts of the isolate ofthe present invention may be used for the treatment and/or prevention ofparasitic infections, preferably N. caninum infections.

Preferably, the extract is selected from the group consisting of liveattenuated parasites, killed and fixed parasites, cell lysates,antigenic polypeptides and polynucleotides encoding antigenicpolypeptides.

Vaccines of the invention may comprise a crude extract of the novelparasitic isolate. Killed and/or chemically fixed parasites or cells canalso be used. Vaccines may also comprise partially or completelypurified polypeptide preparations derived from the parasitic isolate.The polypeptide may be an antigen produced by recombinant DNAtechnology.

In addition, polynucleotide sequences derived from the novel parasiticisolate may be cloned into viruses that transfect host cells in animals.Live attenuated viruses, such as vaccinia or adenovirus, are convenientalternatives to vaccines because they are inexpensive to produce and areeasily transported and administered. It will be appreciated thatpolynucleotides derived from the parasitic isolate of the presentinvention may also be incorporated into non-viral vaccine vectors.

The preparation of vaccines which contain an immunogenic polypeptide(s)as active ingredient(s), is known to one skilled in the art. Typically,such vaccines are prepared as injectables, either as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid prior to injection may also be prepared. The preparation may alsobe emulsified, or the protein encapsulated in liposomes. The activeimmunogenic ingredients are often mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol, or the like and combinations thereof.

In addition, if desired, the vaccine may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, and/or adjuvants which enhance the effectiveness of the vaccine.

Further examples of adjuvants and other agents include aluminumhydroxide, aluminum phosphate, aluminum potassium sulfate (alum),beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions,oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X,Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis,polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A,saponin, liposome, levamisole, DEAE-dextran, blocked copolymers or othersynthetic adjuvants. Such adjuvants are available commercially fromvarious sources, for example, Freund's Incomplete Adjuvant and CompleteAdjuvant (Difco Laboratories, Detroit, Mich.).

Typically, adjuvants such as Quil A, AMPHIGEN™, ALHYDROLGEL™, or amixture of AMPHIGEN™ and ALHYDROGEL™ are used. Other immunostimulatorymolecules such as cytokines (for example, γ-interferon) may also beused.

The proportion of immunogen and adjuvant can be varied over a broadrange so long as both are present in effective amounts. For example,aluminum hydroxide can be present in an amount of about 0.5% of thevaccine mixture (Al₂O₃ basis). Conveniently, the vaccines are formulatedto contain a final concentration of immunogen in the range of from 0.2to 200 μg/ml, preferably 5 to 50 μg/Ml, most preferably 15 μg/ml.

After formulation, the vaccine may be incorporated into a sterilecontainer which is then sealed and stored at a low temperature, forexample 4° C., or it may be freeze-dried. Lyophilisation permitslong-term storage in a stabilised form.

The vaccines are conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkylene glycols or triglycerides; such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1% to 2%. Oral formulations include suchnormally employed excipients as, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate, and the like. These compositions takethe form of solutions, suspensions, tablets, pills, capsules, sustainedrelease formulations or powders and contain 10% to 95% of activeingredient, preferably 25% to 70%. Where the vaccine composition islyophilised, the lyophilised material may be reconstituted prior toadministration, e.g. as a suspension. Reconstitution is preferablyeffected in buffer.

Capsules, tablets and pills for oral administration to a patient may beprovided with an enteric coating comprising, for example, Eudragit “S”,Eudragit “L”, cellulose acetate, cellulose acetate phthalate orhydroxypropylmethyl cellulose.

Vaccine compositions of the present invention may be administered toanimals susceptible to or otherwise at risk of infection to elicit animmune response against the parasite and thus enhance the animals ownimmune response capabilities. Such an amount is defined to be an“immunogenically effective amount”. In this use, the precise amountdepends on the judgement of the prescribing veterinarian or doctor andwould include consideration of the patient's state of health and weight,the mode of administration, the nature of the formulation, and the like.

A variety of vaccination regimes may be effective in immunising cattleand other animals. Preferably, female cattle are vaccinated just priorto or at the time of breeding so as to prevent abortion and reduce thepossibility of congenital infections. A second immunisation may be givenat other time periods before and during gestation. Calves and adultmales may also be vaccinated, if desired. Animals that have previouslybeen exposed to N. caninum or have received colostral antibodies fromthe mother may require booster injections. The booster injection ispreferably timed to coincide with times of maximal challenge and/or riskof abortion.

In order that the present invention may be more clearly understoodpreferred forms will be described with reference to the followingexamples and drawings.

EXAMPLE 1 Isolation of Bovine Neospora sp. in New South Wales

The isolation and characterisation of a Neospora parasite from aclinically normal neonatal Australian Holstein-Friesian calf isdescribed. The calf was born from a dam maintained in a commercial dairyherd of Holstein-Friesian cattle located on the south coast of New SouthWales (NSW) Australia, near Nowra. This herd has been endemicallyinfected with neosporosis for a number of years and has been studiedextensively (Atkinson et al. 2000b). The dam was first identified asbeing serologically positive with an IFAT titre of 1280 for N. caninumin March 1995, having previously aborted in January 1994 but not since.The calf, which was seropositive to N. caninum antigen by westernblotting, remained healthy and showed no clinical signs of N. caninuminfection prior to it's euthanasia (NSW Agriculture) with intravenouspotentiated pentabarbitone (EUTHETAL™) via jugular puncture with a 21gauge needle and 20 ml syringe, dosed to effect. On cessation of life,the brain and spinal cord (including thoracic segments T10 to T15) wereremoved following dissection of the tissues of the skull and cervicalregion and exposure of the central nervous system (CNS) using a saw andbone cutters. With the exception of a 3 cm diameter segment of leftfrontal cerebral cortex which was fixed in 10% formalin for histologicalprocessing, the CNS tissues were placed immediately in antibiotic salinesolution (0.9% NaCl containing penicillin and streptomycin) andhomogenised. Trypsin was then added to 0.05% and the homogenateincubated for 30 mm at 37° C. The homogenate was centrifuged for 10 mmat 1200 g and resuspended in antibiotic saline. The pellet was washedtwice in antibiotic saline with centrifugation.

For tissue culture, the pellet was resuspended in 8 ml of tissue culturemedium and placed using aseptic technique into a flask containing amonolayer of vero cells (Barber et al. 1995). Unfortunately the culturesbecame contaminated and were discarded.

Homogenates in antibiotic saline were injected intraperitoneally intointerferon-gamma receptor knockout (IFN-γRKO, 129/Sv) mice with a 23gauge needle. Mice were injected with homogenate from either infectedbrain (6 mice) or spinal cord extract (6 mice) and then monitored dailyfor clinical signs of disease. Clinical signs suggestive of neosporosisincluding ruffled coat, huddled lethargic appearance and weight loss(Atkinson et al. 1999), were observed in both groups of mice (brain andspinal cord injected) within 23 days post-inoculation with homogenate.Mice were euthanased with carbon dioxide and their brains removed andprocessed as above for infection of tissue culture cells with bovinebrain tissue, however using smaller volumes. Brain homogenates were alsopassaged back into naive IFN-γRKO knock-out mice, which developedclinical signs of neosporosis by 23 days post infection. A third passagein mice resulted in clinical signs being observed from 10 days postinfection.

Histopathological examinations, performed by NSW Agriculture, ofmultiple sections of cerebrum from the clinically normal calf identifiedvery mild multifocal non-suppurative encephalitis characterised by rarefoci of gliosis and perivascular cuffing. No parasitic elements wereobserved, however, the lesions were considered consistent with very mildintrauterine cerebral infection with N. caninum (Boulton et al. 1995).Histological examination of cerebrum from 5 mice inoculated with calfbrain homogenates identified mild multifocal non-suppurativeencephalitis in all 5, with 2 apicomplexan structures resembling N.caninum tachyzoites identified in the cerebral choriod tissue section ofa single mouse. Immunohistochemistry performed on this sectionidentified positive immunoperoxidase staining with anti-Neosporaantisera.

Vero cells infected with brain homogenate from one of these miceresulted in the proliferation of tachyzoites 14 days post infection. Thein vitro growth of these parasites has now been successfully achievedfor 15 months. The isolate was identified as a member of the genusNeospora for the reasons outlined below and was named Nc-Nowra, afterthe geographical location of the study herd.

EXAMPLE 2 Characterisation of Isolated Organisms

Electron microscopy. Tachyzoites were recovered from in vitro cultureand examined by transmission electron microscopy. Tachyzoites were fixedin 2.5% glutaraldehyde, 2% paraformaldehyde in phosphate buffer pH 7.2for 2 hrs; washed three times with 0.1M cacodylate buffer and post-fixedin 1% aqueous osmium tetroxide. After excess fixative was removed bywashing with water, parasites were dehydrated in ethanol, transferred toanhydrous acetone and infiltrated and embedded in Epon resin. The blockswere cut and ultrathin sections examined with a JEM 1010 transmissionelectron microscope. Tachyzoites were identified as N. caninum by virtueof their size, shape and internal structures including the presence ofcharacteristic intracellular organelles such as electron dense rhoptries(Speer et al. 1999).

Western blotting. Tachyzoite lysates of Nc-Nowra, Nc-Liverpool and theME49 strain of T. gondii were analysed by western blotting using seraobtained from mice acutely infected with Nc-Liverpool (Atkinson et al.1999). The antigen profile detected for Nc-Nowra was similar, but notidentical to, that obtained from Nc-Liverpool. The antigen profilepossessed no similarity to that shown by T. gondii.

PCR and DNA sequencing. PCR amplification of Nc-Nowra genomic DNA wasperformed using DNA prepared by standard techniques, from in-vitrocultured tachyzoites. The primers Tim3 and Tim11 were used and theamplification products were analysed by agarose gel electrophoresis(Payne and Ellis, 1996). PCR specifically amplified an internaltranscribed spacer (ITS1) sequence, which was similar in size to thatobtained from Nc-Liverpool DNA. The PCR product was purified using aQIAGEN™ purification kit and the DNA sequenced by cycle sequencing usingprimers Tim3 and Tim11. A consensus sequence derived for the ITS1 ofNc-Nowra was 99% similar to that previously reported for N. caninum(AF029702 and AF038860 for example).

EXAMPLE 3 Nc-Nowra Tachyzoites Used in a Serological Test such as anELISA

A dairy herd, located in the Southern Highlands of NSW, contained 177Friesians with a history of abortion, with 37 abortions being recordedover 18 months spanning 1999/2000. There were two dogs and numerousfoxes on this property, and N. caninum was previously diagnosed on theproperty by post mortem examination of aborted foetuses by NSWAgriculture. The herd is regularly and fully vaccinated againstleptosporidiosis. The dairy herd was bleed for routine investigation ofthe cause of the abortions and these sera were screened for antibodiesto Nc-Nowra using an indirect ELISA protocol developed at UTS anddescribed below.

Nc-Nowra tachyzoites were recovered from in vitro culture and reduced toprotein extracts by resuspension in lysis buffer (20 mM Tris pH 7.5, 150mM NaCl, 1% TRITON™ X-100, 1 mM PMSF, 2 mM DTT, 1 mM benzimidine HCl)and disruption by sonication at 50W/20 KHz for 10-20 secs. The resultingpreparation was dialysed against PBS overnight at 4° C. with one changeof buffer. Protein concentration was determined using the Bradforddye-binding assay (Biorad). NUNC™ 96 well plates were coated withantigen diluted to 1 μg/well in carbonate buffer at 4°C. overnight.Plates were washed three times with PBS/0.03% TWEEN™, and the testbovine serum diluted in ELISA buffer 2 (0.3% TWEEN™, 0.05% bovinehaemoglobin) was added to wells. The sera used were from dairy cattlewhich were prone to abortion. Plates were again incubated overnight at4° C. and then washed three times with PBS/0.03% TWEEN™. Anti-bovineIgG-alkaline phosphatase conjugate was diluted 1:6,000 in buffer 2 andplates were incubated at 37° C. for 3 hrs. Plates were washed withPBS/0.03% TWEEN™ and 1 mg/ml of p-nitrophenylphosphate in carbonatebuffer was added. Plates were incubated at 37° C. for 3 min andabsorbance at 405 nm read in a Biorad ELISA plate reader. A positiveserum was identified as being 2 standard deviations greater than themean negative O.D. result (from 20 sera).

Results. Nc-Nowra crude lysate was used in an ELISA to screen a dairyherd suspected of containing N. caninum positive cows. A graph showing asummary of the O.D. readings obtained from the herd ELISA is in FIG. 1.Four (4) of 177 (2.3%) cows in the herd were seropositive to N. caninumusing this assay. There was a high correlation between ELISA absorbancesobtained using the Nc-Nowra ELISA and the results from a commercial N.caninum ELISA kit, which detected 16 of 177 (9%) cows as seropositivewithin the herd. The commercial kit is also based on a crude lysateantigen, however the NC-1 N. caninum isolate is used. This was isolatedin the U.S.A. and has different biological properties to the Nc-Nowraisolate.

EXAMPLE 4 A Cell Extract of Nc-Nowra Used to Induce an Immune Responsein an Animal

Thirty (30) QS mice were divided equally into 10 groups, each of whichreceived one of the following treatments, 4 weeks apart:

GROUP TREATMENT 1. None 2. Freund's incomplete adjuvant (FIA) alone 3.FIA + Nc-Nowra

Two (2) weeks after the second injection, mice were killed by CO₂asphyxiation, bled and their spleens removed for a cell proliferationassay.

An Nc-Nowra extract was made in the following way and used for eitherinjection into animals or for ELISA. Nc-Nowra tachyzoites were recoveredfrom in vitro culture and reduced to protein extracts by resuspension inlysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mMPMSF, 2 ml DTT, 1 mM benzimidine HCl) and disruption by sonication at50W/20 Hz for 10-20 secs. The resulting preparation was dialysed againstPBS overnight at 4° C. with one change of buffer. Protein concentrationwas determined using the Bradford dye-binding assay (Biorad).

Sera, made by standard procedures, was assayed by ELISA using thefollowing method. NUNC™ 96 well plates were coated with Nc-nowra antigenextract diluted to 1 μg/well in carbonate buffer at 4° C. overnight.Plates were washed three times with PBS/0.03% TWEEN™ and mouse serumdiluted in ELISA buffer 2 (0.3% TWEEN™, 0.05% bovine haemoglobin) wasadded to wells. Serum used was from mice in the treatment groups asdescribed above. Plates were again incubated overnight at 4° C. and thenwashed three times with PBS/0.03% TWEEN™. Anti-mouse IgG-alkalinephosphatase conjugate was diluted 1:6,000 in buffer 2 and plates wereincubated at 37° C. for 3 hrs. Plates were washed with PBS/0.03% TWEEN™and 1 mg/ml of p-nitrophenyiphosphate in carbonate buffer was added.Plates were incubated at 37° C. for 30 mm and absorbance at 405 nm readin a Biorad ELISA plate reader.

Cell proliferation assays were performed on mouse spleen cells asfollows. Spleens were individually placed in wash medium (DMEMcontaining penicillin and streptomycin, DMEM(PS), and forced through a70 μm nylon disposable sieve to yield a single cell suspension. Thecells were concentrated by centrifugation and resuspended in 2 mls redcell lysis buffer and left at room temp. for 1 min. The lysis buffer wasdiluted out with more DMEM/PS, centrifuged and the pellets resuspendedin fresh DMEM/PS for counting. 1×10⁴ cells per well of each suspensionwere added in triplicate to wells of a microtitre plate. Stimulatingantigen at a concentration of 10 μg/well for Con A and Nc-Nowra lysatewas added to the cells. A blank (containing no cells) was included. Thecells were incubated at 37° C. in 5% CO₂ for 4 days. BrdU was then addedand the cells incubated for a further 4 hours. The cells were pelletedto the bottom of the microtitre plate by centrifugation, thesupernatants collected for cytokine assay, and the cells dried andfixed. BrdU incorporation was then detected using a commercialimmunoassay kit (Roche) encompassing an anti-BrdU-POD conjugate.

Results. An increase in absorbance was observed in the serum samplestaken at each time point indicating an IgG response was raised in miceagainst a crude antigen extract of Nc-Nowra. This response wassignificantly greater than that seen in uninjected mice or mice injectedwith adjuvant alone. The time course showed a significant IgG responseat and after 6 weeks post injection in the mice receiving Nc-Nowraextract.

A crude extract of Nc-Nowra, when used as a stimulating antigen, alsostimulated cell proliferation in spleen cells from mice, showing a cellmediated immune response was induced.

EXAMPLE 5 Nc-Nowra is Only Mildly Pathogenic in the Mouse

A single experiment was performed to investigate the pathogenicity ofNc-Nowra in the BALB/C mouse. This model has been used previously toinvestigate the pathogenicity of N. caninum (Nc-Liverpool and Nc-SweB1)in vivo (Atkinson et al. 1999). Groups of female mice were injectedintraperitoneally with either saline, 10⁶ (Nc-Liverpool or Nc-Nowra) or10⁴ (Nc-Nowra) tachyzoites grown in-vitro. Mice were weighed daily andeuthanased when clinical signs dictated (to alleviate unnecessarysuffering) or at the end of the experiment at day 31 post-infection. Thebrains of all mice in the experiment were removed, fixed in 10% formalinand examined pathologically.

Results. All mice injected with saline survived until the end of theexperiment and gained weight steadily over this time period. Mice givenNc-Liverpool rapidly became ruffled and lethargic and euthanasia ofaffected mice began at day 20 post infection. By day 31, only 4 out of 9mice remained in this group. Mice given Nc-Nowra were not as rapidlyaffected with 7 out of 17 mice surviving until day 31 post infection.

Histological sections from 3 of the mice injected with saline failed toidentify significant histological lesions (no further mice were examinedin this group). Lesions of varying severity and characterised asmultifocal non-suppurative to necrotising encephalitis were identifiedin 5 of 8 mice receiving 10⁴ Nc-Nowra, all of 9 mice receiving 10⁶Nc-Nowra and all of 9 mice receiving 10⁶ Nc-Liverpool. Lesions weregraded in severity as per previous studies (Atkinson et al. 1999) with 4of the mice receiving 10⁴ Nc-Nowra, 4 of the mice receiving 10⁶ Nc-Nowraand all 9 mice of the 10⁶ Nc-Liverpool mice, judged to have moderate tosevere lesions. These findings show that, like Nc-SweB1, which was alsoisolated from the bovine (Stenlund et al. 1997), Nc-Nowra is lesspathogenic in the BALB/C mouse, compared to Nc-Liverpool.

Five (5) BALB/C mice injected i.p. with Nc-Nowra infected brain from aknock-out mouse were maintained in a healthy condition for 7 months, andshowed no clinical signs of neosporosis. These mice seroconverted to N.caninum antigen (by western blotting). After euthanasia, analysis ofbrain pathology showed mild brain lesions consistent with N. caninuminfection. No parasitic stages were seen during the pathologic analyses.

EXAMPLE 6 A Live Infection by Nc-Nowra can Prevent Neosporosis in MiceInfected with Nc-Liverpool

Tachyzoites of the Nc-Nowra isolate were harvested from an in vitroculture of Vero cells and diluted in 0.9% saline to a concentration of10⁵/ml. Ten BALB/c mice were infected subcutaneously with 10⁴tachyzoites per mouse. A further fifteen mice were injected with 0.9%saline only. Three weeks post infection the ten Nc-Nowra infected miceand ten of the saline injected mice were infected with 10⁶ Nc-Liverpooltachyzoites recovered from tissue culture. The five remaining salineinjected mice were injected with saline again. The mice were monitoreddaily for clinical signs (ruffled and lethargic) and weighed daily fromday 13 p.i. Mice were euthanased after losing 20% of their body weight.Blood was collected from all mice and brains from a selection from eachgroup. The blood was allowed to clot, spun and the serum removed. Thebrains were fixed in 10% formalin and sent to NSW Agriculture forpathologic examination.

Results

Group 1: injected with saline and challenged with Nc-Liverpool (positivecontrol, n=10):

Clinical signs were observed in the Nc-Liverpool only infected mice onday 14 p.i. and euthanasia of mice started on day 21. All ten mice inthis group died between day 21 and day 25 p.i.

Group 2: infected with Nc-Nowra and challenged with Nc-Liverpool (n=10):

In contrast, only 2 mice in the Nc-Nowra/Nc-Liverpool infected micedeveloped clinical signs and these were euthanased on days 19 and 20.Both of these mice were small and light in weight for their age at thebeginning of the experiment. Such mice normally succumb quickly toneosporosis and were included as an internal positive control. All eightother mice in this group remained healthy throughout the experiment andshowed no signs normally associated with N. caninum infection such asweight loss, head tilting or motor discoordination. It was concludedthat the Nc-Nowra infection had induced protective immunity against theclinical signs of neosporosis associated with Nc-Liverpool infection.

Group 3: injected with saline and given no parasite challenge (negativecontrol, n=5)

All mice in this group remained healthy.

Brain Pathology

Five (5) micron sections were cut from brains, stained with H and E andexamined for evidence of brain lesions. Lesions were graded on a scaleof 0 to 5 with 5 being the most severe type of lesion. The average scorefor the Nc-Liverpool infected brains (Group 1) examined was 3.5 whilethe average score for the Nc-Nowra/Nc-Liverpool (Group 2) infectedbrains examined was 1.5. It was concluded that the prior Nc-Nowrainfection had significantly reduced brain lesion score in thisexperiment.

EXAMPLE 7 Prior Infection by Nc-Nowra has no Effect on Litter Size inthe Pregnant QS Mouse

Five (5) week-old, female QS mice were injected subcutaneously with 10⁴in-vitro derived tachyzoites of Nc-Nowra. Three weeks later ovulation inall mice was synchronised by two hormone injections (PMSG and hCG) given48 hours apart. At day 8 of pregnancy 1 of the treatment groups wasgiven 10⁶ tachyzoites of Nc-Liverpool. All mice were killed at day 14 ofpregnancy and the uterine implantation sites were analysed for viablefoetuses.

Results: The table below summarises the treatment groups and the meanlitter sizes obtained. No significant difference in mean litter size wasfound between the treatment groups, indicating Nc-Nowra does not causefoetal loss in this model.

Number of mice Treatment Mean Litter Size (S.E.) in the group None 15.2(1.18) 23 Nc-Nowra 16.9 (1.73) 12 Nc-Nowra/Nc-Liverpool 17.3 (1.45) 19Sera from all dams killed at day 14 were assayed for IgG to N. caninumantigen using the ELISA method described in Example 4. Mice infectedwith Nc-Nowra showed high levels of IgG to N. caninum, whereas mice inthe other two groups did not (FIG. 2).

EXAMPLE 9 Infection of Pregnant QS Mice with Nc-Nowra

Nine (9) week-old female QS mice were mated following synchronisation ofovulation with 2 hormone injections (PMSG and hCG) 48 hrs apart. At day8 of gestation mice were given either a subcutaneous injection of saline(control group), 1×10⁴ or 1×10⁶ Nc-Nowra tachyzoites derived from tissueculture. All mice were euthanased at day 14 of gestation and uterineimplantation sites were analysed for viable and non-viable foetuses.Statistical analyses was performed using an Analysis of Variance.

Total nucleic acid was made from the excised foetal tissue by standardmethods involving lysis in buffer containing SDS, EDTA and proteinase K,followed by phenol chloroform extraction and ethanol preciptation. TheseDNAs were analysed for the presence of N. caninum DNA by PCR usingprimers that were designed to be specific for N. caninum from thesequence of the ITS1 reported previously (Payne and Elis, 1996). The PCRreaction was heated at 95° C. for 2 min, then subject to 35 cycles of95° C. 45 sec, 50° C. 45 sec and 72° C. for 2 min. The reaction was thentreated at 72° C. for 5 min to allow primer extension to be complete.

Results: The table below summarises the results of this experiment.Injection of mice with Nc-Nowra at day 8 of gestation caused no clinicalsigns of disease and no significant foetal loss (p=0.3355).

Group Mean % of resorptions/litter (S.E.) Saline 6.813 ± 3.535 1 × 10⁴Nc-Nowra 26.75 ± 24.43 1 × 10⁸ Nc-Nowra 7.243 ± 3.626

No N. caninum-specific PCR product was obtained from these foetal DNAs,showing no evidence for N. caninium infection at day 14 of gestation.The positive control (N. caninum DNA) gave the required band of approx.400 bp. This study does not exclude the possibility that N. caninum maybe detected in older foetuses or pups using a similar experimentaldesign with Nc-Nowra.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed in Australia before thepriority date of each claim of this application.

REFERENCES

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1. A Neospora caninum isolate isolated from the central nervous system of a dairy calf, the isolate having AGAL Accession No. NM01/22338, and wherein the isolate is naturally attenuated in its ability to cause neosporosis in a laboratory animal.
 2. An isolated host cell infected with the Neospora caninum isolate as claimed in claim
 1. 3. The host cell as claimed in claim 2 having AGAL Accession No. NM01/22339.
 4. A vaccine composition comprising the Neospora caninum isolate as claimed in claim
 1. 5. A method for inducing protective immunity against neosporosis caused by Neospora caninum in cattle or mice, the method comprising administering to the cattle or the mice the vaccine composition as claimed in claim
 4. 6. The method as claimed in claim 5, wherein the protective immunity is induced in a cow.
 7. A vaccine composition comprising the isolate as claimed in claim 1, wherein the isolate is in the form of a live isolate.
 8. A method of detecting IgG antibodies to the Neospora caninum isolate of claim 1 in a serum sample from cattle or mice infected with said isolate comprising assaying the serum sample for IgG antibodies to the Neospora caninum isolate by ELISA. 